Cytochrome c is an essential component for mediating electron transfer between the primary dehydrogenases and the terminal oxidase for the oxidation of substrate with reduction of molecular oxygen to H2O. This electron transfer reaction is based on an oxidation-reduction of the heme iron. Recently attempts have been made to apply the electron transfer reaction of cytochrome c as new materials imitating biological materials or elements, namely biochips; e.g. by using cytochrome c552 of Hydrogenobacter thermophilus (Kodama et al., U.S. Pat. No. 5,459,046). Acetic acid bacteria including Gluconobacter and Acetobacter possess highly efficient ability for sugar- and sugar alcohol-oxidation and are industrially used for producing vinegar and L-sorbose which is used as intermediate of vitamin C production. In oxidative fermentation, cytochrome c plays an important role to complete the oxidation. Cytochrome c proteins have been purified and characterized from many organisms, including Gluconobacter; e.g. Matsushita et al. reported the purification of CO-binding cytochrome c553 (CO) (molecular weight, 48 kDa) from Gluconobacter suboxydans (FEMS Microbiol. Lett., 10:267-270, 1981) and later the cytochrome c553 (CO) was found to be identical to the second subunit of alcohol dehydrogenase of Gluconobacter. Amplification of the cytochrome c553 (CO) in an alcohol dehydrogenase second subuniteficient Gluconobacter slightly improved L-sorbose production from D-sorbitol in its specific rate (g-product per g-cell-hour) as disclosed in J. Ferment. Bioeng., 74, 209-213, 1992 (Y. Takeda et al.). In addition to the cytochrome c553, cytochrome c551(AL) (molecular weight 55 kDa) and cytochrome c551,(CO) (molecular weight 72 kDa) [Ameyama et al., Agri. Biol. Chem. 51, 2943-2950 (1987)] were also isolated from Gluconobacter. The cytochrome c551(AL) is one of the subunits of aldehyde dehydrogenase of Gluconobacter suboxydans consisting of two subunits; the other subunit is primary aldehyde dehydrogenase of 86 kDa. AL in cytochrome c551(AL) stands for xe2x80x9caldehydexe2x80x9d.
This invention provides DNA sequences and polynucleotides which correspond to a newly isolated cytochrome c551 that is one of the essential components mediating electron transfer between the primary dehydrogenase and the terminal oxidase. Accordingly, part of this invention is a polynucleotide comprising a DNA sequence selected from the group consisting of SEQ ID No. 1, a DNA sequence which is capable of hybridizing to SEQ ID No. 1, and a DNA sequence which encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by SEQ ID No. 2 or encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by a DNA sequence which is capable of hybridizing to SEQ ID No. 1.
A DNA sequence which is capable of hybridizing to SEQ ID No. 1 includes a sequence which is completely complementary to SEQ ID No. 1, and also includes sequences which hybridize under standard stringency conditions which are described in more detail below. Additional polynucleotides of this invention encode the cytochrome c551 polypeptides and the fusion proteins described immediately below.
Also part of this invention are the cytochrome c551 polypeptides. These include a polypeptide which is encoded by any of the above polynucleotide and a cytochrome c551 which has the following characteristics: a molecular weight of about 18-20 kD by gel filtration and about 17-19 kD on SDS-PAGE, which when reduced has an alpha absorption maximum at 551 nm, a beta absorption maximum at 522 nm, and a gamma absorption maximum at 417 nm, which contains about 1 mole of heme per mole of protein, and which has an isoelectric point of about 3.95. Also part of this invention is a cytochrome c551 which has the following characteristics: a molecular weight of about 18-20 kD by gel filtration and about 15.8-17.8 kD on SDS-PAGE, which when reduced has an alpha absorption maximum at 551 nm, a beta absorption maximum at 522 nm, and a gamma absorption maximum at 417 nm, which contains about 1 mole of heme per mole of protein, and which has an isoelectric point of about 3.75. These polypeptides may further include one, two, or all three of the specific amino acid sequences SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5. Also included are active fragments of the above polypeptides.
This invention includes a fusion protein which comprises any polypeptide of this invention, fused with an enzyme, preferably an alcohol aldehyde dehydrogenase.
Also part of this invention is a vector suitable for expression in a pro- or eukaryotic host cell comprising any polynucleotide of this invention, preferably a DNA sequence selected from the group consisting of SEQ ID No. 1, a DNA sequence which is capable of hybridizing to SEQ ID No. 1, and a DNA sequence which encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by SEQ ID No. 2 or encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by a DNA sequence which is capable of hybridizing to SEQ ID No. 1.
Host cells containing polynucleotides of this invention are contemplated, particularly the polynucleotide described immediately above. A host cell may be transformed by an expression vector containing the selected polynucleotide, or may have the polynucleotide integrated into its genome.
Such host cells may be of eukaryotic origin, preferably a mammalian or plant cell, or may be of prokaryotic origin. These host cells may in particular be obtained from bacteria, such as Escherichia coli, Pseudomonas putida, Acetobacter xylinum, Acetobacter pasteurianus, Acetobacter aceti, Acetobacter hansenii, and Gluconobacter oxydans, preferably Gluconobacter oxydans DSM No. 4025.
This invention is also directed to a process for producing cytochrome c551, which comprises cultivating a host cell of this invention, particularly a host cell containing the preferred DNA sequence, in an appropriate culture medium and recovering the cytochrome c551 from the culture medium.